Candida albicans is a human pathogen which causes mild diseases such as vaginitis and thrush in patients with normal immune systems, but which is life-threatening in immuno-deficient patients. With the increasing use of chemotherapy in cancer treatment, immuno-compromised patients have become more common. The usual therapy for systemic candidal infections is amphotericin B, a polyene antibiotic. However, this drug concentrates in the liver and can cause damage, especially in patients on chemotherapy and in the elderly. The need for improved therapy has spurred research for new anticandidal drugs. A preferred source for completely novel structures with antibiotic activity is fermentation broths of microorganisms found in the wild, as well as other natural products. The classical method for screening these products is the disc diffusion test in which the broth is placed in a well or on a paper disc and allowed to diffuse into the solid growth medium seeded with the test organism. After growth, potential antibiotics are revealed by a zone of killing surrounding the disc. The size of the zone is determined both by the concentration and efficacy of the antibiotic. The classical test has several limitations for detecting new drugs. Since this test has been used for years by many pharmaceutical companies, most, if not all, of the easily detected antibiotics such as polyenes have been isolated. If a good drug is produced in very low quantities, it can be missed in the conventional assay. Even with increased sensitivity, the diffusion assay cannot discriminate between agents that are toxic for humans and those that are specific for fungi. Thus, a primary goal of screen development is sensitive and specific assays. One powerful tool for devising sensitive screens is manipulating the test organism genetically. For example, Selitrennikoff has published a screen for fungal cell wall inhibiting agents based upon the unique behavior of a mutant (os-1) of the fungus Neurospora crassa [Selitrennikoff, Anti. Micro. Agents and Chemo., 23, 727, (1983)]. This kind of screen is of great interest because it is unlikely that agents specific for fungal cell walls would be harmful to humans. Other specific screens using mutant test organisms are in use. There are significant differences in cell wall structure and physiology between Candida albicans and fungi with well-characterized genetic systems such as Neurospora crassa. This makes development of means for genetic manipulation of Candida highly desirable. Genetic manipulations of Candida albicans have been hampered by several factors:
1. It is difficult to obtain recessive mutations due to the diploid nature of the organism.
2. There is no sexual cycle for recombination.
3. The parasexual cycle is laborious, requiring protoplast fusion of suitably marked auxotrophic strains.
4. No method for introduction of DNA either as plasmid vectors or linear DNA fragments has been found.
After the discovery that DNA could be reliably introduced into the bakers yeast Saccharomyces cerevisiae by plasmid vectors and linear DNA, transformation systems for other fungi seemed feasible. With the appropriate vector/host pair, genes from diverse sources such as bacteria, fungi and mammalian cells have been cloned and expressed in Saccharomyces. Mutations have been introduced with plasmids by gene disruption and by in vitro mutagenesis. Very high levels of normal yeast enzymes have been obtained by cloning yeast genes on high copy number plasmids and hybrid gene products have been produced by fusion of yeast DNA sequences with foreign DNA such as the bacterial gene for .beta.-galactosidase. Therefore, transformation provides a method for introducing a wide variety of genetic alterations into an organism.
Development of a transformation system for Candida albicans has been difficult because a suitable vector/host system has not been available. The vector must have a selectable marker because transformation generally occurs at a low frequency and detection would otherwise be unacceptably laborious. Two types of selectable markers are known: genes conferring resistance to an antibiotic to a sensitive host, and wild-type genes complementing auxotrophic mutations in the recipient host. Due to the natural resistance of Candida to most antibiotics, the complementation approach is more feasible. The Candida gene for orotidine-5'-phosphate decarboxylase has recently been isolated by complementation of an auxotrophic mutant in a heterologous host (A. Gillum, E. Tsay, and D. R. Kirsch, Mol. Gen. Genetics, 198(1) pgs 179-182 (1984)). However, it was not possible to isolate the proper Candida host strain with the equivalent mutation.